Ab72996

Cultures were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Primary antibodies against NFH (Abcam, ab72996; 1∶1000), synapsin-I (Millipore, AB1543P; 1∶100), activated caspase-3 (BioVision, 3015–100; 1∶100) and phosphorylated (S473) Akt (Abcam ab66138; 1∶200) were diluted in blocking solution (5% donkey serum, 1 mg/ml BSA IgG and protease free, in PBS) and incubated overnight at 4°C. Samples were incubated with species-specific secondary antibodies (Jackson Immunoresearch) for 2 h at room temperature. TMR-conjugated α-bungarotoxin (Sigma, T0195) staining for evidence of acetylcholine receptor (AChR) clusters was performed at 1 µg/ml in PBS for 15 min prior to cell permeabilization and primary antibody incubation. For visualization of nuclei in myotubes, Hoechst 33258 (1 µg/ml) or DAPI was used for live or fixed samples, respectively. After the staining protocol was completed, MFC was peeled from the dish by gently pulling it from the proximal to distal side, to minimize severing of axons. Prolong mounting medium was added and covered with a #1.5, 18×18 mm coverslide.

MN cultures were washed with warm 1X PBS and immediately fixed in 4% PFA for 15 minutes. Samples were permeabilized in 0.5% triton for 10 minutes and blocked in blocking solution (1 mg/mL BSA; 10% Goat serum; 0.1% triton in PBS) for 1 hour at room temperature. Samples were incubated with primary antibodies in blocking solution for 12 hours at 4 °C. Antibodies were used in the following concentrations: Elavl2 1:50 (Rabbit; Proteintech, 14008-1-AP), NFH 1:500 (Chicken; Abcam, ab72996) 1:1,000 MAP2 (Rabbit; Millipore, AB5622), 1:100 Tau (Mouse; Abcam, ab80569). Samples were then incubated for 2 hours with fluorescent secondary antibodies: DyLight 405 anti-chicken 1:200; Alexafluor 488 anti-rabbit 1:500; AlexaFluor 647 anti-mouse 1:500; AlexaFluor 594 anti-rabbit 1:500. Samples were mounted using ProLong^® gold antifade reagent.

GC was excised from P60 mice and cleared of connective tissue, washed in PBS, fixed in 4% PFA, washed once more, and then incubated with 1 g/ml Rhodamine Red-Conjugated Bungarotoxin (Sigma-Aldrich). Tissues were washed and then treated with methanol at −20°C for 5 min, washed, and then blocked in blocking solution for 1 h. Tissues were then rocked with appropriate primary antibodies diluted in blocking solution at room temperature overnight. Antibodies were used at the following concentrations: anti-Neurofilament Heavy Chain 1:500 (NFH, Abcam, ab72996; 1:1000), synaptophysin (Millipore, MAB5258; 1:300), synaptotagmin (Alomone Labs, ant-003; 1:300), anti-NRP1 (1:100), anti-Sema3A (1:100), anti-NRP2 (1:100), and anti-Sema3B (1:100). After having been washed, secondary antibodies (DyLight 405 anti-chicken 1:500; AlexaFluor-488 anti-chicken 1:500; AlexaFluor-647 anti-rabbit 1:500) were added for 4 h at room temperature. Muscle fibers were spread into monolayers under a stereomicroscope and affixed to slides using VectaShield (Vector Laboratories). Cover slides were sealed with clear nail polish.